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metabolism in cultured cells, especially in primary cultured neurons which are rich in complex gangliosides. In contrast with the current paradigm in medicinal chemistry, we applied few substances of defined structure to complex cell culture systems. Primary cell cultures are artificial compared with multicellular organisms

[41], but are sufficiently complex compared with cell lines or enzyme assays. As will become clear below, the results observed would have not been obtained in enzyme assays.

Because the target compounds can be expected to interfere with the metabolism of endogenous ceramide, we investigated the effect of the ceramide analogs 11 and 12 on incorporation of biosynthetic precursors L-serine and D-galactose into ceramide-containing lipids of primarily cultured murine cerebellar granule cells. Serine-incorporation reflects de-novo biosynthesis of sphingolipids. On the other hand, galactose is, in part after intracellular metabolism to other carbohydrates, enzymatically added to ceramide derived from the de-novo pool and from the salvage pool, which can contribute up to 80% of glycosphingolipid biosynthesis. Exo-genously added ceramide analogs can also be metabolically labeled by galactose

[42]. In brief [43, 44], primary neurons were prepared from the cerebellum of 6-day-old mice and the cells were treated with different concentrations of the target compounds for 24 h. Radiolabeled 3-[14C]serine or [14C]galactose was added to the culture medium and incorporation into newly synthesized sphingolipids was analyzed after labeling for 24 h. Lipids were extracted, separated by thin-layer chro-matography, and visualized with a phosphoimager. Radioactivity found in the selected lipids is expressed in relation to untreated cells.

L-[3-14C]serine labeling of sphingolipids in the presence of 11 led to a concentration-dependent decrease of sphingomyelin and glucosylceramide levels whereas the amount of ceramide increased. Unexpectedly, [14C]galactose labeling indicated inhibition of sialyltransferase II (SAT II, GD3-synthase) at much lower concentrations of 11 of 10 mM in the culture medium and, at higher concentrations, also of GalNAc-transferase (GM2-synthase, Figure 1.4.7). Even more exciting results were obtained with 12. As determined by [14C]galactose labeling, concentrations of 10 ^m in the culture medium caused dramatic - up to 40-fold -elevations in the levels of LacCer, GM3, and GD3, which indicated inhibition of GM2-synthase. These findings were confirmed by metabolic labeling with L-[3-14C]serine and [4,5-3H]sphinganine. The compound with opposite stereochemistry at the carbon bearing the alkyl chain had nearly no effect, indicating specificity.

To determine whether 12 inhibits GalNAc-transferase directly, we applied 100 ^m 12 in an enzyme assay with the homogenate of insect Sf21-cells over expressing murine GalNAc-transferase [45]. No inhibition was detected, so we had to conclude that 12 operates indirectly, either after metabolism, or by interfering with intra-cellular membrane transport in the same way as, for example, the macrolide brefeldin A [46]. Brefeldin A specifically binds to a usually transient intermediate of the reaction between an ARF-protein (ARF = ADP-ribosylating factor), guanosine diphosphate, and the Sec-7 domain of a guanine-nucleotide-exchange factor (GEF). This disturbs the activation cycle of ARF and exocytotic membrane flow is interrupted [47]. Because other ceramide analogs not mentioned here interfere with

a-series

GlcCer, GlqS1Cer; LacCer, Gal^1,4Glc^1Cer; GM3, NeuAca2,3GaljS1,4GlcjS1Cer; GD3, NeuAca2,8NeuAca2,3GaljS1,4GlcjS1Cer; GA2, GalNAcjS1,4GaljS1,4GlcjS1Cer; GM2, GalNAcjS1,4(NeuAca2,3)GaljS1,4GlcjS1Cer; GD2, GalNAqSM(NeuAca2,8NeuAca2,3)-GaljS1,4GlcjS1Cer. The next a-series ganglioside would be ganglioside GM1 (Figure 1.4.1).

O-series

Fig. 1.4.7. Early steps in combinatorial ganglioside biosynthesis [9]. The sites of (indirect) inhibition by 11 and 12 are indicated. Abbreviations: Cer, ceramide, N-acylsphingosine; Glc, glucose; Gal, galactose; GalNAc, N-acetylgalactosamine; NeuAc, N-acetylneuraminic acid; SAT, sialyltransferase; GalNAcT, N-acetylgalactosaminyltransferase;

a-series

GlcCer, GlqS1Cer; LacCer, Gal^1,4Glc^1Cer; GM3, NeuAca2,3GaljS1,4GlcjS1Cer; GD3, NeuAca2,8NeuAca2,3GaljS1,4GlcjS1Cer; GA2, GalNAcjS1,4GaljS1,4GlcjS1Cer; GM2, GalNAcjS1,4(NeuAca2,3)GaljS1,4GlcjS1Cer; GD2, GalNAqSM(NeuAca2,8NeuAca2,3)-GaljS1,4GlcjS1Cer. The next a-series ganglioside would be ganglioside GM1 (Figure 1.4.1).

other glycosyltransferases, we do not consider 12 as a brefeldin mimic. The results with galactose labeling were qualitatively confirmed by incorporation of l-[3-14C]serine and [4,5-3H]sphinganine in the sphingolipids in the presence of 12 and its diastereomer. Analysis of other ceramide analogs indicates that several glyco-syltransferases within the ganglioside biosynthesis pathway can be inhibited by this approach [10].

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